Studies proposed in this application encompass and extend long-standing interests of the Principal Investigator in the cell and molecular biology of blood and vascular cells. The proposed Program is comprised of two ambitious, impactful projects designed to fill important conceptual gaps in their respective fields, and that have long been judged to be of interest and relevance to the overall mission of the NHLBI; namely (1) The role of PECAM-1 in vascular cell function, and (2) The pathophysiology of neonatal alloimmune thrombocytopenia (NAIT). PECAM-1 (also known as CD31) is a cellular adhesion and signaling receptor that functions in circulating blood cells to limit the rate and extent of cellular activation. PECAM-1 is also the most highly-expressed component of the endothelial cell-cell junction, where it functions as a homophilic adhesive stress-response protein to maintain endothelial cell junctional integrity and speed restoration of the vascular permeability barrier following inflammatory or thrombotic challenge. We will exploit our recent determination of the three-dimensional structure of the PECAM-1 homophilic binding domain, our development of innovative PECAM-1-targeted tools and animal models, and our discovery that PECAM-1 is subject to conformational affinity modulation to examine the potential for PECAM-1 to serve as a novel therapeutic target for a wide range of vascular permeability disorders, including sepsis-induced vascular leakage and ischemia-reperfusion injury. This Program will also focus on developing new tools, models and treatments for NAIT ? a rare, but catastrophic, clinically important bleeding disorder caused by maternal antibodies generated in response to paternally-inherited antigens present on fetal platelets that re-cross the placenta and bind to fetal and/or neonatal platelets, resulting in thrombocytopenia often serious enough to require transfusion, and in the most severe cases causing intracranial hemorrhage and intrauterine death. Despite advances in treatment, NAIT remains the leading cause of intracranial hemorrhage in full-term infants, often leading to lifelong disability. We propose to combine recent transformative advances in CRISPR gene editing technology with the ability to generate megakaryocyte progenitor cells, megakaryocytes, and platelets from induced pluripotent stem cells to establish a transformative diagnostic platform designed to narrow the existing ?diagnostic gap? to improve treatment and care of NAIT - namely the creation of platelet alloantigen-specific cell lines capable of long-term self-renewal, cryopreservation, and distribution; thereby providing a potentially inexhaustible source of iPS-derived platelets for diagnostic, investigative, (and potentially future therapeutic) use. CRISPR technology has also allowed us to develop a novel humanized mouse model of NAIT that will allow us to resolve a series of outstanding issues in platelet alloimmunity. Taken together, this research program will apply cutting-edge technology to lay the groundwork for continuing advances in the diagnosis and treatment of these and related blood and vascular disorders of relevance to the mission of the National Heart, Lung and Blood Institute.